Skip Navigation

Cardiovascular Research 2007 76(3):517-527; doi:10.1016/j.cardiores.2007.08.007
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Díez, M.
Right arrow Articles by Peinado, V. I.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Díez, M.
Right arrow Articles by Peinado, V. I.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2007, European Society of Cardiology

Plasticity of CD133+ cells: Role in pulmonary vascular remodeling

Marta Díez, Joan A. Barberà, Elisabet Ferrer, Raquel Fernàndez-Lloris, Sandra Pizarro, Josep Roca and Victor I. Peinado*

Department of Pulmonary Medicine, Hospital Clínic-Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Ciber de Enfermedades Respiratorias, Universitat de Barcelona, Barcelona, Spain

*Corresponding author. Servei de Pneumologia, Hospital Clínic. Villarroel, 170, 08036 Barcelona, Spain. Tel.: +34 93 2275540; fax: +34 93 2275455. vpeinado{at}clinic.ub.es

Received 2 May 2007; revised 1 August 2007; accepted 15 August 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Objective Studies in pulmonary arteries (PA) of patients with chronic obstructive pulmonary disease (COPD) suggest that bone marrow-derived endothelial progenitor cells (CD133+) may infiltrate the intima and differentiate into smooth muscle cells (SMC). This study aimed to evaluate the plasticity of CD133+ cells to differentiate into SMC and endothelial cells (EC) in both cell culture and human isolated PA.

Methods Plasticity of granulocyte-colony stimulator factor (G-CSF)-mobilized peripheral blood CD133+ cells was assessed in co-cultures with primary lines of human PA endothelial cells (PAEC) or SMC (PASMC) and in isolated human PA. We also evaluated if the phenotype of differentiated progenitor cells was acquired by fusion or differentiation.

Results The in vitro studies demonstrated CD133+ cells may acquire the morphology and phenotype of the cells they were co-cultured with. CD133+ cells co-incubated with human isolated PA were able to migrate into the intima and differentiate into SMC. Progenitor cell differentiation was produced without fusion with mature cells.

Conclusions We provide evidence of plasticity of CD133+ cells to differentiate into both endothelial cells and SMC, reinforcing the idea of their potential role in the remodeling process of PA in COPD. This process was conducted by transdifferentiation and not by cell fusion.

KEYWORDS Stem cells; Pulmonary circulation; Hypertension; Cell differentiation; Endothelial function


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Patients with COPD show striking changes in pulmonary muscular arteries and precapillary vessels that explain the presence of pulmonary hypertension [1,2]. The hallmark of pulmonary vascular derangement in this group of patients is the thickening of the intimal layer due to smooth muscle-like cell proliferation. The etiology of vascular changes at the initial stage of the disease remains still uncertain. The most plausible hypotheses about the origin of intimal cells include: a) transdifferentiation of endothelial cells into smooth muscle cells [3,4]; b) differentiation of adventitial fibroblasts into myofibroblasts and migration into the intimal layer throughout the media [5,6]; and, c) recruitment of circulating bone-marrow stem cells throughout endothelium, migration towards the intima and differentiation into smooth muscle cells [7]. The latter is specially suggestive because it does not exclude any of the aforementioned hypotheses since the differentiation process of stem cells may be sensitive to different microenvironments. Moreover, it provides a simple explanation about the degree of cell maturity, proliferative capacity and regional heterogeneity within cells observed in both the media and intimal layers along the vascular tree [8–10].

The identification of stem cells playing a role in vascular repair remains unresolved. There is consensus on the existence of a circulating endothelial precursor with properties similar to those of embryonic angioblasts [11]. This precursor, with a bone-marrow origin, can be identified by a characteristic surface phenotype positive for CD34, CD133, and VEGF receptor-2 (VEGFR-2) [12] and can differentiate to endothelial cell in vitro [13]. Recent studies have suggested that CD133+ cells may possess differentiation potential, both in vitro and in vivo, not limited to endothelial lineage [14], although it is not clear whether this apparent plasticity could be attributed in all tissues to transdifferentiation [14] or cell fusion processes [15]. Although there is not doubt that these cells may play a role in tissue repair, given the potential of CD133+ cells in terms of plasticity, they might also participate in the progression or maintenance of pre-existing lesions [7,16]. In the pulmonary circulation, this hypothesis is supported by a recent study in COPD showing an increased number of CD133+ cells infiltrating the hyperplasic intima of pulmonary arteries (PA), very close to denuded areas of endothelium [17]. In that study, the number of progenitor cells attached to the endothelium correlated with the thickness of the arterial wall, suggesting a potential association with the severity of the remodeling process. A potential link between systemic inflammation and circulating progenitors has also been suggested in COPD [18].

We hypothesized that CD133+ cells possess ability to differentiate into smooth muscle cells (SMC) in the intima of pulmonary arteries, hence potentially contributing to initiate and/or perpetuate vessel remodeling. Accordingly, the aim of this study was to investigate the plasticity of human hematopoietic CD133+ cells to differentiate in vitro into both pulmonary atery smooth muscle cells (PASMC) and pulmonary artery endothelial cells (PAEC). In a second set of experiments, we evaluated the potential of CD133+ cells to migrate into the intima and differentiate into SMC incubating CD133+ cells into the lumen of human explanted PA. Finally, we explored the mechanisms, fusion or transdifferentiation, by which CD133+ cells may acquire the phenotype of tissue cells in human PA.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
2.1 Cell primary cultures and characterization
The human hematopoietic progenitor cell line CD133+ was obtained from Cambrex Bio Science (Walkersville, MD). Before using, CD133+ cells were expanded for 10 days in flasks at a density of 1x105 cells/mL in stem span medium supplemented with Flt-3ligand (100 ng/ml), StemCell factor (100 ng/ml) and Thrombopoietin (100 ng/ml) (Stem Cell Technologies, Vancouver, Canada) at 37 °C in 5% CO2.

The PAEC line was purchased from American Type Culture Collection (HPAE-26, ATCC, Manassas, VA). Cells were cultured in endothelial cell growth medium (ECGM) composed of F-12K nutrient mixture (GIBCO BRL, Grand Island, NY) supplemented with 10% FBS, penicillin/streptomycin (100 U/ml), heparin (0.1 mg/ml) and endothelial cell growth factor (0.03 mg/ml).

The PASMC line was obtained from Cambrex Bio Science. Cells were cultured in smooth muscle cell growth medium (SMCGM) composed of SMC basal medium (Clonetics, Cambrex) supplemented with 5% FBS, insulin, human fibroblast growth factor (hFGF-B), gentamicin/amphotericin-B and human epidermal growth factor (hEGF) (SmGM-2 SingleQuots, Clonetics, Cambrex).

After expansion, the hematopoietic progenitor cell phenotype was assessed by flow cytometry for the expression of CD133 and by immunofluorescence for the expression of CD45, CD133, CD34, CD31, VEGF2, vWF and vimentin markers and by RT-PCR.

The phenotype of PAEC was characterized by the LDL-uptake, affinity to Ulex europaeus agglutinin I (UEA), and by the positive or negative expression of vimentin, vWF, CD34, CD133, CD45 and {alpha}-actin. The expression of genes related with the endothelial linage, CD34, vWF, vimentin, and eNOS were also determined by RT-PCR.

The phenotype of PASMC was characterized morphologically and by the immunoreactivity to antibodies against smooth muscle {alpha}-actin, and desmin. The expression of genes related with SMC, SMC {alpha}-actin and the potassium channels Kv1.5 and BKCa were also determined by RT-PCR.

Further details regarding material and methods can be found in the online supplementary material.

2.2 Labeling of CD133+ cells
For all co-culture experiments with PAEC and PASMC and with explanted PA, human CD133+ cells were previously labeled with 10 µg/ml 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated LDL (Di1-acLDL; Molecular Probes, Invitrogen, Eugene, OR) for 3 h at 37 °C, followed by 3 washes with PBS. The nontransferable dye Dil-acLDL was used for live monitoring of the human CD133+ cells during co-cultivation.

2.3 Co-culture experiments
For co-culture experiments, PAEC or PASMC were cultured in 24-well fibronectin plates (BD Biosciences, San Jose, CA) in ECGM and SMCGM respectively. After 48 h of culture, Dil-acLDL CD133+ cells were added at a density of 4x104 cells/well allowing them to differentiate. After 6 and 12 days, co-cultures were fixed in 4% paraformaldehyde and stained for UEA or SMC {alpha}-actin. At least 5 independent experiments were performed at each experimental condition. Control experiments for the effect of media on differentiation of Dil-acLDL CD133+ cells included co-cultures with PAEC in SMCGM and co-cultures with PASMC in ECGM.

In a second set of experiments, co-cultures were performed using a device (Nunc, Nalge Nunc International, Roskilde, Denmark) that maintained both cell lines separated by a membrane (pore size 0.2 µm) that allows the transit of molecules but not cells. Mature cells, PAEC or PASMC, were cultured in 24-well fibronectin plates for 6 and 12 days as previously described. Labeled progenitor cells were put into device chambers and then inserted inside wells containing mature cells.

2.4 Characterization of differentiated CD133+ cells in co-cultures
Co-cultures of PAEC with Dil-acLDL-labeled CD133+ cells were incubated with FITC-labeled UEA (Sigma-Aldrich) overnight at 4 °C in the dark and examined under fluorescence microscope. Cells positive for both FITC-UEA and Dil-acLDL were judged to be endothelial cells which have differentiated from CD133+ cell. These co-cultures were also incubated with primary antibodies against CD31, vWF and CD34.

To evaluate the differentiation of CD133+ cells into smooth muscle cells, the co-culture of PASMC with Dil-acLDL-labeled CD133+ cells was assessed for the expression of {alpha}-smooth muscle actin and desmin. Also theses co-cultures were assessed for negativity for CD31 and UEA.

Differentiation of Dil-acLDL labeled cells was quantified by flow cytometry and under fluorescence microscope. For flow cytometric analysis, the percentage of double positive cells, and then UEA+/Dil-acLDL-labelled cells, in both types of co-cultures was determined with a FACSCalibur flow cytometer. For two-color flow cytometry, 8x104 cells were incubated with UEA-FITC at 4 °C for 1 h. Then cells were washed in PBS containing 1% heat-inactivated FCS and 0.1% sodium azide. Cells were analyzed with CellQuest software (BD Biosciences, Immunocytometry Systems). Each analysis included at least 5000–10,000 events. A light-scatter gate was set up to eliminate cell debris from the analysis. For fluorescence microscope analysis, double positive cells were counted over a total of 200 cells, and then SMC {alpha}-actin+ filaments/Dil-acLDL-labeled cells for PASMC-CD133+ cell co-cultures or UEA+/Dil-acLDL-labeled cells for PAEC-CD133+ cell co-cultures.

2.5 Cultures of explanted PA with CD133+ cells
Intralobar pulmonary arteries (1 to 1.5 mm diameter) were dissected from human lungs (n=5), obtained at lung resection for bronchogenic carcinoma. From each lung specimen, two 3 cm-long segments of PA were isolated under microscope in a laminar flow hood and cultured in ECGM for 3 h at 37 °C in 5% CO2. Then, Dil-acLDL labeled CD133+ cells were injected using a 2 ml syringe with a 19G needle into the arterial lumen. The ends of arteries remained opened in order to improve gas exchange. The artery with CD133+ cells inside was left in ECGM at 37 °C in 5% CO2 for 2 and 4 days. Five independent experiments were performed. The endothelium of PA was dissected as previously described [17]. Some segments of arteries were fixed in 4% paraformaldehyde, cryoembedded in O.C.T and frozen at –80 °C for immunofluorescence analysis; the remaining ones were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde for transmission electron microscopy (TEM).

Differentiation of CD133+ cells over the endothelial surface was characterized morphologically and by immunofluorescence using antibodies against vimentin, CD31, and CD34. Differentiation of CD133+ cells in the intima was assessed on cryostat sections of artery rings immunostained with an antibody against human {alpha}-SMC actin and by TEM. Under TEM, progenitors cells were identified by the content of LDL into the cytosol, since LDL particles can be seen as homogenous and dense vesicles.

Studies in human lung specimens had been approved by the Institutional Review Board and written informed consent was obtained from the patients before surgery.

2.6 Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) for chromosome X and 15 were used to detect fusioned nuclei in harvested endothelia from pulmonary arteries (n=5). We used commercially available probes labeled with FITC to detect the number of chromosomes X or 15 (Q-biogen Inc, Vista, CA) in interphase nuclei. Paraformaldehyde-fixed endothelia were mounted on poly-L-lysine treated slides. Nuclei with two or more dots were counted in 5 randomly areas and expressed as percentage of total nuclei.

2.7 RT-PCR
Total RNA was extracted from culture cells using a commercially available kit (Trizol Reagent; Life Technologies, GIBCO BRL; Carlsbad, CA).

2.8 Immunohistochemistry
For intercellular proteins, cells were permeabilized with 0.5% Triton X-100 for 30 min except when Dil-AcLDL was used. After blocking with non immune serum for 1 h, we used the following primary mouse monoclonal antibodies: CD45 (1:750 dilution; Novocastra Laboratories Ltd., Burlingame, CA), CD133 (1:10 Miltenyi Biotech, Bergisch Gladbach, Germany), Vimentin (1:1000 dilution; DAKO Cytomation, Carpinteria, CA), CD34 (1:150 dilution; Novocastra Laboratories Ltd), CD31 (1:200 dilution; DAKO Cytomation), VEGFR2 (1:200 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA), vWF (1:150 dilution; Dako Cytomation), Desmin (1:200 dilution; DAKO Cytomation), {alpha}-smooth muscle actin (1:750 dilution; DAKO Cytomation). All incubations were performed overnight at 4 °C in the dark. Then, cells were washed 3 times with PBS and incubated for 1 h at RT with the secondary antibody against mouse conjugated with indocarbocyanine (Cy3) (1:200 dilution; Jackson Immuno Research, West Grove, PA) or conjugated with FITC fluorescein (1:200 dilution; Jackson Immuno Research). After three washes, nuclei were stained for 5 min with DAPI, mounted with a fluorescence mounting media (Vectashield, Vector Laboratories, Burlingame, CA) and visualized by a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
3.1 In vitro differentiation of CD133+ cells
After 12 days of culture, CD133+ cells cultured alone in ECGM (Fig. 1) showed low affinity for the specific endothelial cell marker UEA, although they did not acquire morphology of PAEC. Supplementation with vascular endothelial growth factor (VEGF) or with a cocktail of cytokines improved the signal intensity for UEA, although the size and shape of the cells remained different from mature PAEC (Fig. 1). No differentiation of CD133+ cells into SMC was observed when they were cultured alone with SMCGM.


Figure 1
View larger version (70K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Differentiation of CD133+ cells after 12 days of culture in fibronectin plates. Cells were assessed for the affinity to UEA (green) and the ability of LDL uptake (red). Progenitor cells were cultured with ECGM (a-d), with ECGM supplemented with VEGF (e-h) or with Endocult® medium (i-l). Nuclei were counterstained with DAPI (blue). Merged images (d,h,l). Scale bar, 20 µm.

 
3.2 Co-culture of CD133+ cells with PAEC and PASMC
After 6 days of co-culture with PAEC, many Dil-acLDL labeled cells had integrated with them. These labeled cells displayed significant increases of their cell length and surface area, similar to adjacent mature cells (Fig. 2A). More than 28% of total cells in co-culture were double positive for UEA and Dil-acLDL (about 85% of Dil-acLDL labeled cells) as revealed by FACs (Fig. 3). Rounded mononuclear cells that remained undifferentiated were negative for UEA (data not show). Dil-acLDL labeled cells also differentiated in endothelial cells in this co-culture when they were incubated with SMCGM instead of ECGM.


Figure 2
View larger version (87K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Co-culture of progenitor cells with mature cells. A) Dil-acLDL-labeled CD133+ cells (red) after 12 days of co-culture with PAEC (a). Co-cultures were stained with UEA (green), which is specific for mature endothelial cells (b). Nuclei (blue) were counterstained with DAPI (c). Merged images (d). Scale bar, 20 µm. B) Dil-acLDL-labeled CD133+ cells (red) after 12 days of co-culture with PASMC (e). Co-cultures were immunostained with a monoclonal antibody against SMC {alpha}-actin (green), which is specific for SMC (f). In blue, nuclei were counterstained with DAPI (g). Merged images (h). Scale bar, 20 µm.

 

Figure 3
View larger version (41K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Representative flow cytometric analysis from co-cultures of Dil-acLDL labeled CD133+ cells with PAEC (A) and PASMC (B) after 1, 6 and 12 days. A) After 6 days, more than 28% of Dil-acLDL labeled cells of total cells in co-culture were double positive for UEA and Dil-acLDL. B) Any Dil-acLDL positive cells showed affinity to UEA at day 6 or at day 12.

 
As in co-cultures with PAEC, after 6 days of co-culture with PASMC, integrated Dil-acLDL labeled cells adopted a similar size and shape of adjacent mature cells. Under fluorescence microscope, Dil-acLDL labeled cells expressed SMC proteins such as specific SMC {alpha}-actin (Fig. 2B) and desmin but not CD31 or affinity to UEA. The percentage of Dil-acLDL labeled cells showing {alpha}-actin organized filaments ranged between 8% and 9% of total SMC. Any Dil-acLDL–positive cells in this co-culture showed affinity to UEA neither at day 6 nor at day 12 (Fig. 3) (see online supplementary material). Dil-acLDL labeled cells also differentiated in smooth muscle cells in this co-culture when they were incubated with ECGM instead of SMCGM.

3.3 Co-cultures of human CD133+ cells with mature cells separated by a trans-well membrane
In the double chamber devices, CD133+ cells acquired the phenotype of mature cells cultured in the other side of the membrane, and all cells showed affinity for UEA when co-cultured with endothelial cells and {alpha}-actin when co-cultured with smooth muscle cells. The inverse stain was negative for both cultures. The morphology after 12 days was similar to that observed after 12 days when progenitors were cultured alone (Fig. 4).


Figure 4
View larger version (74K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Co-cultures of Dil-acLDL-labeled progenitor cells with PAEC (A) and PASMC (B) using a device maintaining both cell lines separated by a membrane. b) After 12 days progenitors were stained with FITC-UEA for cells cultured with PAEC or f) stained with SMC {alpha}-actin for cells cultured with PASMC. c,g) Nuclei were stained with DAPI. d,h) Merged images. Scale bar, 20 µm.

 
3.4 Differentiation of CD133+ cells in explanted pulmonary arteries
Fig. 5A shows different sections of a PA incubated with Dil-acLDL labeled CD133+ cells. Two days after injecting CD133+ cells into the arterial lumen, a number of mononuclear cells were localized in the intima. The majority of them preserved the original morphology and remained close to the endothelial surface. After 4 days of co-incubation, cells acquired a more elongated morphology and the majority of them were localized in the intima towards the media.


Figure 5
View larger version (95K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 A) Transverse sections of a human PA co-incubated with CD133+ cells. a) Tranverse section at low magnification of a representative pulmonary artery. Scale bar, 400 µm. b) Control section without progenitor cells. c) An artery section after two days of incubation with progenitors. Many cells, with rounded shape, are infiltrating the intima. d) Transverse section of a PA segment after 4 days of incubation with progenitors. Many cells have migrated through the intima towards the media. The progenitor cell shape changes from round to large and flat. Methylen blue stain. Scale bar in b, c and d, 100 µm. B) Immunofluorescence of a transverse section of a human PA after 4 days of co-incubation with Dil-acLDL-labeled CD133+ cells. a) The red fluorescent tracer identifies the progenitor cells infiltrating the intima b) In green is shown the fluorocrom FITC revealing the immunoreaction against SMC {alpha}-actin. c) The nuclei are stained with DAPI d) merged image showing double positive cells (arrows) indicating differentiation of progenitor cells into smooth muscle cells. Scale bar, 20 µm. C) Immunofluorescence of an endothelial surface of a human PA after 2 days of co-incubation with Dil-acLDL-labeled CD133+ cells. a) The red fluorescent tracer identifies the progenitor cells b) In green is shown the fluorocrom FITC revealing the immunoreaction against vimentin. c) The nuclei are stained with DAPI d) merged image showing double positive cells indicating differentiation of progenitor cells into endothelial cell. Scale bar, 10 µm. D) Transmission electron microscopy of a transverse section of a human PA co-incubated with LDL-labeled progenitor cells. LDL can be identified as dense and homogenous vesicles in the cytoplasm. a) A progenitor cell not yet differentiated showing a great number of LDL vesicles inside cytoplasm (arrows). Scale bar, 2 µm. b) A progenitor, identified by a large cytoplasmic vesicle, is integrating in the endothelium. Scale bar, 5 µm. c) Some progenitors can be identified infiltrating the intima. Scale bar, 5 µm. d) A greater magnification of a progenitor infiltrating the intima touching the elastic internal lamina. In this micrograph showing a longitudinal section of a progenitor cell, the dense bodies (arrows), characteristic of smooth muscle cells, are well preserved. Scale bar, 2 µm.

 
Immunofluorescence studies performed on transverse sections revealed that some Dil-acLDL labeled cells confined into the intima were immunoreactive for smooth muscle {alpha}-actin indicating differentiation into SMC (Fig. 5B). By contrast, Dil-acLDL-labeled cells adhered to the endothelium acquired an endothelial-like morphology and expressed the endothelial cell markers CD31 and vimentin (Fig. 5C).

Sections of pulmonary arteries co-incubated with acLDL-labeled CD133+ cells were examined by electronic microscopy (Fig. 5D). CD133+ cells were identified by the presence of large LDL vesicles in the cytosol. Some labeled cells, attached to the luminal surface, showed endothelial cell morphology; whereas cells located in the intima showed smooth muscle cell-like morphology. Some cells infiltrating the intima showed apparent dense bodies characteristic of smooth muscle cell filaments [19]. Interestingly, acLDL-labeled cells infiltrating the intima were preferentially localized close to denuded areas of endothelium.

3.5 Cell fusion evaluation
ImmunoFISH analysis revealed the presence of some scattered polyploid nuclei in pulmonary endothelia (Fig. 6A) (see online supplementary material). The presence of tetraploid nuclei was not uniform over the extension analyzed. The number of fusioned nuclei ranged between 5 and 10% indicating that fusion was not a rare event. We also performed immunoFISH looking for polyploid nuclei on endothelial layers after incubation with Dil-acLDL labeled CD133+ cells, as described in the preceding section. In this case, we did not find fusioned nuclei within labeled CD133+ cells indicating that the mature phenotype was acquired by transdifferentiation (Fig. 6B). Moreover, in electronic microscope studies we did not observe membrane fusions between progenitor and mature cells, further supporting the findings of in situ hybridization.


Figure 6
View larger version (61K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Immunofish of human male PA endothelium. In situ hybridization was performed using a probe to identify chromosome X. A) Normal endothelium of a PA. a) In green (FITC) is shown the chromosome X of nuclei in interphase. b) The cytoskeleton of endothelial cells was revealed using an antibody against vimentin and revealed with a secondary antibody linked to the fluorochrom Cy3. c) Nuclei were stained with DAPI. The fusion of two nuclei can be observed in the image. d) Merged images. Some nuclei show more than one copy of chromosome X indicating that they are polyploid. Scale bar, 15 µm. B) Progenitor cells differentiating on the endothelium of a PA. e) Green dots on nuclei identify the chromosome X. The cytoskeleton of cells was revealed using an antibody against vimentin and revealed with a secondary antibody linked to the fluorochrom FITC. f) The red fluorescent tracer identifies the progenitor cells g) Nuclei are stained with DAPI. h) Merged images. None of the nuclei show more than one copy of chromosome X indicating that there is not fusion between cells during differentiation. Scale bar, 15 µm.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 Acknowledgments
 References
 
In a previous study [17] we observed an increased number of CD133+ cells in PA of COPD patients. Positive cells were not only confined to the endothelial surface but also identified in the vessel wall, thus suggesting that these cells could play a role in pulmonary vessel remodeling. In this report, performed in vitro and in explanted human PA, we provide evidence of the plasticity of CD133+ cells to differentiate into both endothelial and smooth muscle cells.

The differentiation process of bone-marrow derived vascular progenitor cells was mimicked with the simplest scenario found in the arterial wall by co-culturing adult human PASMC or PAEC with CD133+ cells. In these experiments, the fate of CD133+ cells was conditioned by the type of mature cell they were co-cultured with, evidencing the plasticity of CD133+. Evidence of the multipotentiality of CD133+ cells has been previously provided by Torrente and co-workers [14] who demonstrated their differentiation into skeletal muscle cells when co-cultured in the presence of a feeder layer of myogenic cells.

Presumably, mature cells in co-culture may act as a source of cytokines and growth factors that modulate the differentiation process. This was deduced from co-cultures of progenitor and mature cells, separated by a trans-well membrane (pore size 0.2 µm). In this setting, CD133+ cells acquired markers of mature cell when they were incubated separately from PAEC or PASMC. Yet, these factors were insufficient to induce morphological changes, hence showing the crucial role of cell-to-cell contact for morphology acquisition. At present, we have not identified the molecules, namely growth factors and cytokines, which may participate in the mechanisms of differentiation in vitro or in vivo. Previous studies have shown that common vascular progenitor cells may give rise to endothelial cells in vitro upon exposure to supplemental VEGF [13,20,21], and to SMCs when treated with platelet derived growth factor-BB (PDGF-BB) [22]. In these experiments, the formation of early outgrowth endothelial colonies is produced after 8–9 days of incubation whereas in our studies, full cell differentiation was already apparent at day 6 of co-culture. In this respect, we also observed that isolated CD133+ cells incubated with a supplement of VEGF differentiated into endothelial-like cells after 12 days. We observed a better signal intensity for endothelial cell markers when cells were incubated with a cocktail of cytokines, suggesting that complementary co-stimulation with other growth factors, a condition closer to the physiological environment, may accelerate the effect of VEGF on endothelial differentiation.

After checking in vitro the capability of CD133+ cells to differentiate into both endothelial and smooth muscle cell types, we aimed to explore the dynamics of spontaneous cell differentiation in co-cultures of human CD133+ injected into the lumen of explanted human PA. In this setting, mature cells of the endothelium or the intima might provide the micro-environment that could be crucial in determining the fate of CD133+ cells. Initially we selected a PA from a patient with severe COPD. That PA showed a prominent intima and denudated endothelium. When progenitor cells were co-incubated with the PA, we expected to find many progenitor cells repopulating the endothelium. However, a number of CD133+ cells migrated through the intima towards the media (Fig. 5A). During the 4 days of co-incubation, a gradual change in morphology from round to large and flat shape was observed in labeled cells, indicating that progenitor cells undergo trans-differentiation into smooth muscle-like cells. This was further supported by the identification of SMC {alpha}-actin expression. Furthermore, electron microscope analysis revealed a few progenitor cells, identified by the presence of LDL vesicles in the cytoplasm, fully integrated within cells of the media. All together, these results show that CD133+ cells are capable to migrate from the lumen into the intima and differentiate into SMC, at least in remodeled PA from COPD. We also incubated progenitor cells with PA with a more preserved endothelium. Interestingly, in these cases only few cells migrated into the intima. CD133+ cells integrated within the endothelium acquired endothelial cell morphology (Fig. 5D). In the other hand, cells that migrated into the intima were close to endothelial fenestrations indicating that CD133+ cells might have reached the intima through these areas and that endothelium may operate in two ways, releasing local mediators for cell differentiation and as a physical barrier to cell migration towards the intima. In these respect, our experiments confirm that endothelial cell damage or endothelial denudation is crucial for the progression of pulmonary vascular remodeling in COPD [17,23]. This observation is in line with evidences showing that contribution of recruited hematopoietic stem cells to vascular remodeling is strongly enhanced by tissue injury [24–26].

It has been demonstrated that adult bone marrow cells in culture can be driven to differentiate into many different cell types, including hepatocytes, cardiomyocytes, skeletal muscle and neurons. However, there has been considerable controversy regarding their contribution to non-hematopoietic tissues in vivo. In fact, adult bone marrow cells might not be able to transdifferentiate autonomously, but instead this might depend on cell fusion to achieve the differentiated state [27,28]. We also wondered whether circulating CD133+ cells can regenerate tissues in vivo by fusion with differentiated cells or if they might differentiate into mature cells without cell contact with other mature cells. For this reason, we first screened pulmonary endothelium for polyploid nuclei in order to check whether fusioned cells might arise spontaneously in normal tissue. In situ hybridization revealed the presence of scattered polyploid nuclei (Fig. 6A) in PA endothelium, indicating that fusion is not a rare event in endothelial cells. However, cell fusion appears to be restricted to mature cells since in co-cultures or when progenitors were co-incubated with explanted PA we did not observe the intermediate formation of multi-nucleated cells (Figs. 5C and 6BGo). Moreover, in PA, some progenitor cells differentiated alone, without apparent contact with other cells. Despite we have not found multinucleated cells during differentiation of progenitors, we cannot rule out the possibility that in vivo the two processes may occur together. In either case, the mature cell seems to provide a direction or orientation that is necessary to restore the injured tissue.

In summary, our data demonstrate that CD133+ cells are capable of committing themselves to more than one lineage given the right environmental cues. This means that CD133+ cells lying in the luminal surface of pulmonary arteries can differentiate into endothelial cells or they can migrate into the intima and differentiate into smooth muscle cells. In pulmonary arteries, growth factors embedded in the extracellular matrix and/or the contact with resident cells might exert a strong signal to cell differentiation into smooth muscle cell or endothelial cell. Such differentiation seems to be conducted without evidence of cell fusion. Exploration of the growth factors and adhesion molecules that underlie the linkage between CD133+ differentiation and intimal thickening in injured arteries is important to better understand the mechanisms of vascular repair and, perhaps, may lead to novel approaches for the prevention of vascular remodeling.

Time for primary review 34 days


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
Acknowledgments
 References
 
Appendix A Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2007.08.007.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
References
 
We thank Blanca Reyes and Isabel Crespo for their expert technical assistance. The authors also thank the Servei Científic Tècnic from the Universitat de Barcelona, specially to Nuria Cortadellas and Almudena García, for their assistance in TEM studies. This work has been supported by grants FIS 03/0549 and 06/0360, SEPAR-2005, MTV 04-310 and EU IP-018725 (Pulmotension). The group is integrated into the cibeRes (ISCIII CB06/06).


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 

  1. Wilkinson M., Langhorne C.A., Heath D., Barer G.R., Howard P. A pathophysiological study of 10 cases of hypoxic cor pulmonale. Q J Med (1988) 66:65–85.[Web of Science][Medline]
  2. Wright J.L., Petty T., Thurlbeck W.M. Analysis of the structure of the muscular pulmonary arteries in patients with pulmonary hypertension and COPD: national Institutes of Health nocturnal oxygen therapy trial. Lung (1992) 170:109–124.[Web of Science][Medline]
  3. Arciniegas E., Ponce L., Hartt Y., Graterol A., Carlini R.G. Intimal thickening involves transdifferentiation of embryonic endothelial cells. Anat Rec (2000) 258:47–57.[CrossRef][Medline]
  4. Frid M.G., Kale V.A., Stenmark K.R. Mature vascular endothelium can give rise to smooth muscle cells via endothelial-mesenchymal transdifferentiation: in vitro analysis. Circ Res (2002) 90:1189–1196.[Abstract/Free Full Text]
  5. Sartore S., Chiavegato A., Faggin E., Franch R., Puato M., Ausoni S., et al. Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant. Circ Res (2001) 89:1111–1121.[Abstract/Free Full Text]
  6. Shi Y., O'Brien J.E., Fard A., Mannion J.D., Wang D., Zalewski A. Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries. Circulation (1996) 94:1655–1664.[Abstract/Free Full Text]
  7. Sata M. Circulating vascular progenitor cells contribute to vascular repair, remodeling, and lesion formation. Trends Cardiovasc Med (2003) 13:249–253.[CrossRef][Web of Science][Medline]
  8. Archer S.L., Huang J.M., Reeve H.L., Hampl V., Tolarova S., Michelakis E., et al. Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ Res (1996) 78:431–442.[Abstract/Free Full Text]
  9. Frid M.G., Aldashev A.A., Dempsey E.C., Stenmark K.R. Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities. Circ Res (1997) 81:940–952.[Abstract/Free Full Text]
  10. Frid M.G., Aldashev A.A., Nemenoff R.A., Higashito R., Westcott J.Y., Stenmark K.R. Subendothelial cells from normal bovine arteries exhibit autonomous growth and constitutively activated intracellular signaling. Arterioscler Thromb Vasc Biol (1999) 19:2884–2893.[Abstract/Free Full Text]
  11. Caprioli A., Jaffredo T., Gautier R., Dubourg C., Dieterlen-Lievre F. Blood-borne seeding by hematopoietic and endothelial precursors from the allantois. Proc Natl Acad Sci U S A (1998) 95:1641–1646.[Abstract/Free Full Text]
  12. Peichev M., Naiyer A.J., Pereira D., Zhu Z., Lane W.J., Williams M., et al. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood (2000) 95:952–958.[Abstract/Free Full Text]
  13. Gehling U.M., Ergun S., Schumacher U., Wagener C., Pantel K., Otte M., et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood (2000) 95:3106–3112.[Abstract/Free Full Text]
  14. Torrente Y., Belicchi M., Sampaolesi M., Pisati F., Meregalli M., D'Antona G., et al. Human circulating AC133(+) stem cells restore dystrophin expression and ameliorate function in dystrophic skeletal muscle. J Clin Invest (2004) 114:182–195.[CrossRef][Web of Science][Medline]
  15. Terada N., Hamazaki T., Oka M., Hoki M., Mastalerz D.M., Nakano Y., et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature (2002) 416:542–545.[CrossRef][Medline]
  16. Hayashida K., Fujita J., Miyake Y., Kawada H., Ando K., Ogawa S., et al. Bone marrow-derived cells contribute to pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension. Chest (2005) 127:1793–1798.[CrossRef][Web of Science][Medline]
  17. Peinado V.I., Ramírez J., Roca J., Rodriguez-Roisin R., Barberà J.A. Identification of vascular progenitor cells in pulmonary arteries of patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol (2006) 34:257–263.[Abstract/Free Full Text]
  18. Palange P., Testa U., Huertas A., Calabro L., Antonucci R., Petrucci E., et al. Circulating haemopoietic and endothelial progenitor cells are decreased in COPD. Eur Respir J (2006) 27:529–541.[Abstract/Free Full Text]
  19. Cross P.C., Mercer K.L., Freeman W.H., et al. Cell and tissue ultrastructure. A functional perspective. In: (1993) 95–121.
  20. Kanayasu-Toyoda T., Yamaguchi T., Oshizawa T., Hayakawa T. CD31 (PECAM-1)-bright cells derived from AC133-positive cells in human peripheral blood as endothelial-precursor cells. J Cell Physiol (2003) 195:119–129.[CrossRef][Web of Science][Medline]
  21. Lin Y., Weisdorf D.J., Solovey A., Hebbel R.P. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest (2000) 105:71–77.[Web of Science][Medline]
  22. Shoji M., Sata M., Fukuda D., Tanaka K., Sato T., Iso Y., et al. Temporal and spatial characterization of cellular constituents during neointimal hyperplasia after vascular injury: potential contribution of bone-marrow-derived progenitors to arterial remodeling. Cardiovasc Pathol (2004) 13:306–312.[CrossRef][Web of Science][Medline]
  23. Peinado V.I., Barberà J.A., Ramírez J., Gómez F.P., Roca J., Jover L., et al. Endothelial dysfunction in pulmonary arteries of patients with mild COPD. Am J Physiol, Lung Cell Mol Physiol (1998) 18:L908–L913.[Web of Science]
  24. Gill M., Dias S., Hattori K., Rivera M.L., Hicklin D., Witte L., et al. Vascular trauma induces rapid but transient mobilization of VEGFR2(+)AC133(+) endothelial precursor cells. Circ Res (2001) 88:167–174.[Abstract/Free Full Text]
  25. Lee S.H., Wolf P.L., Escudero R., Deutsch R., Jamieson S.W., Thistlethwaite P.A. Early expression of angiogenesis factors in acute myocardial ischemia and infarction. N Engl J Med (2000) 342:626–633.[Abstract/Free Full Text]
  26. Sata M., Saiura A., Kunisato A., Tojo A., Okada S., Tokuhisa T., et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med (2002) 8:403–409.[CrossRef][Web of Science][Medline]
  27. Balsam L.B., Wagers A.J., Christensen J.L., Kofidis T., Weissman I.L., Robbins R.C. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature (2004) 428:668–673.[CrossRef][Medline]
  28. Wagers A.J., Sherwood R.I., Christensen J.L., Weissman I.L. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science (2002) 297:2256–2259.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 ChestHome page
V. I. Peinado, S. Pizarro, and J. A. Barbera
Pulmonary Vascular Involvement in COPD
Chest, October 1, 2008; 134(4): 808 - 814.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Díez, M.
Right arrow Articles by Peinado, V. I.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Díez, M.
Right arrow Articles by Peinado, V. I.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?